Folliculotropin releasing hormone

ABSTRACT

A method for producing the Folliculotropin Releasing Hormone (FRH) biosynthetically is provided by utilizing, as key starting materials, fresh hypothalamic tissue, a buffered incubation medium, an ATP synthesizing system, radioactively labeled amino acids and a mixture of 21 naturally occuring amino acids. Biosynthesis of the FRH is accomplished by incubation of the hypothalamic tissue for a short period of time in the presence of all the reagents necessary to promote the biosynthetic system to produce the Folliculotropin Releasing Hormone (FRH) of the hypothalamus gland of mammals. Examples of methods to isolate the FRH free of the luteotropin releasing hormone (LRH) are provided.

This invention relates to the Folliculotropin Releasing Hormone (FRH) ofthe hypothalamus of mammals, and to alternative methods of biosynthesisand isolation of said FRH. This biosynthetic FRH has the biological,chemical and hormonal properties of the Folliculotropin ReleasingHormone (FRH) of the hypothalamus in the brain of mammals including man.

BACKGROUND OF THE INVENTION

A substance that stimulates the secretion of the follicle stimulatinghormone has been reported by many investigators. This substance ispresent in homogenates of hypothalamic tissue from many species. Thehypothalamus is a part of the forebrain; prosencephalon, which unlikethe cerebellum and crebral hemispheres, has maintained a relativeconstancy of arrangement throughout its phylogenetic history. Thehypothalamus is a small section of tissue of the diencephalon which isthe posterior part of the prosencephalon, consisting of thehypothalamus, thalamus, metathalamus, and epithalamus. The hypothalamusis not considered as an organ with boundaries, but rather as a region ofbrain tissue which has significant functions in many aspects ofmammalian physiology. Basically, the hypothalamus is divisible intomedial and lateral portions. The medial portion joins the thirdventricle. The lateral portion contains cells that are diffuselyarranged among the fibers of what has long been called the medialforebrain bundle. An anatomic relationship which is a constant featureof the hypothalamus is its intimate association with the pituitarygland. The pituitary or hypophysis consists of anterior and posteriorlobes, and the pars intermedia. Biochemical transport of theneurohormones from the hypothalamus to the anterior lobe oradenohypophysis is provided by certain blood vessels in a portal systemwhich is a network of capillaries. The blood in these capillaries passesdown the pituitary stalk and becomes distributed through another systemof capillaries in the anterior lobe and one of the neurohormones thustransported is folliculotropin releasing hormone (FRH).

It is generally considered that there is one hypothalamic releasinghormone for each anterior pituitary hormone. This concept has not beenproven and it has been found that the known luteotropin releasinghormone (LRH) and thyrotropin releasing hormone (TRH) stimulate therelease of two pituitary hormones. It has been found that TRH canstimulate the release of prolactin and thyrotropin and that LRH canstimulate the release of luteotropin and folliculotropin.

A prolactin releasing hormone (PRH) has recently been described as beingchemically distinct from TRH. [Valverde-R., et al., Endocrinology, 91,982 (1972)]. We now describe the biosynthesis of a FolliculotropinReleasing Hormone (FRH) chemically separable from LRH.

It has been proposed by Schally and White [Schally, et al., Science,173, 1036 (1971) and White, Hypophysiotropic Hormones of theHypothalamus, J. Meites, ed., p. 248, 1970] that luteotropin releasinghormone (LRH) is the natural regulator of both luteotropin andfolliculotropin. More recently, [Guillemin and Burgus, ScientificAmerican, 227, 24 (1972)] it has been stated by noted investigators that"A third gonadotropic hormone, FSH (follicle stimulating hormone), mayhave its own hypothalamic releasing factor, FRF, but that has not beendemonstrated."

We have found, indeed, that LRH releases folliculotropin to a limiteddegree in addition to a potent release of luteotropin. More important isour finding that hypothalamic extracts contain a hormone, chemicallydistinct from LRH, that stimulates a very high level of folliculotropinrelease as well as a limited release of luteotropin. The functions ofthe gonads are thus controlled by two hypothalamic releasing hormones,LRH and FRH, that both regulate the release of luteotropin andfolliculotropin from the anterior pituitary. Each releasing hormoneappears to have a primary and a secondary regulatory activity.

Only a few nanograms of each releasing hormone appears to exist in thehypothalamus of a single animal. Therefore, hypothalami from thousandsof animals are required to isolate a sufficient quantity for structureelucidation studies. The biosynthetic approach to the isolation of a newhormone makes it possible to attach a radioactive tag to the compound ofinterest and increase the amount of hormone present in the startingtissue. Once the hypothalamic hormone has been radioactively labeled, itcan be added to a large amount of tissue. This process increases thetotal quantity of hormone present to a quantity suitable for structureelucidation after the necessary purification procedures.

THE INVENTION

It has been found now that the folliculotropin releasing hormone (FRH)is chemically distinct from other releasing hormones present inhypothalamic extracts. The folliculotropin releasing hormone can bebiosynthesized and radioactively labeled by a process involvingincubation of fresh hypothalamic tissue in the presence of a radioactiveamino acid.

The folliculotropin releasing hormone (FRH) activity present inhypothalamic extracts has become discernable since the structureelucidation [Matsuo, et al. Biochem. Biophys. Res. Commun., 43, 1334(1971)) and synthesis (Sievertsson, et al., Biochem. Biophys. Res.Commun., 44, 1566 (1971) and others] of the luteotropin releasinghormone (LRH).

Through careful study of the behavior of synthetic LRH in numerousbiological test systems, it has been shown that LRH can stimulate therelease of luteotropin and folliculotropin. Synthetic LRH will notrelease, in vitro, more than 35,000 ng/ml of FSH even at dose levels upto 500 ng/ml. A dose of 0.9 ng and 500 ng have similar releasingactivity for folliculotropin. Our partially purified hypothalamicextracts at Stage SP have shown extremely potent FolliculotropinReleasing Activity (FRH). Dose levels as low as 0.2 hypothalamicfragment equivalent (h.f.e.) from some preparations released > 128,000ng FSH/ml in the in vitro assay procedure. Folliculotropin ReleasingHormone (FRH) activity can be separated from Luteotropin ReleasingHormone (LRH) activity by chromatographic procedures described ingreater detail herein below. For example:

Incubated tissue is lyophilized, extracted and defatted to give astandard preparation (Stage SP). Stage SP material is twice passedthrough Bio-Gel P-2 (Stage P-2) and then chromatographed on a Sephadexpartition column in solvent system A. After this procedure the LRH andFRH activities are separated. A second chromatography in the same systemgave a good separation of areas corresponding to the known LRH and thepreviously unknown FRH.

Alternatively, Stage P-2 Folliculotropin Releasing Hormone (FRH)material was chromatographed on a Sephadex partition column in solventsystem B. The PC-B FRH was then chromatographed ondiethylaminoethyl-Sephadex. The LRH and FRH present are cleanlyseparated, LRH is unretained and FRH is eluted with 500mM NH₄ OAc aftera continuous or stepwise gradient.

Alternatively, Stage PC-B FRH was chromatographed oncarboxymethyl-Sephadex. The FRH active material is unretained and theLRH active material is eluted with 100 mM NH₄ OAc after a stepwisegradient.

Alternatively, Stage PC-A Folliculotropin Releasing Hormone (FRH) waschromatographed on carboxymethyl-Sephadex. The FRH present wasunretained and the LRH remaining was eluted with 100 mM NH₄ OAc after astepwise gradient.

Alternatively, Stage PC-A Folliculotropin Releasing Hormone (FRH) waschromatographed on carboxymethylcellulose. The FRH present wasunretained and the LRH present was eluted with 70 mM NH₄ OAc during acontinuous gradient from 2 mM to 100 mM NH₄ OAc.

Alternatively, PC-A FRH and LRH was chromatographed ondiethylaminoethyl-Sephadex. The LRH material present is unretained andthe Folliculotropin Releasing Hormone (FRH) is eluted with 500 mM NH₄OAc after a continuous or stepwise gradient from 2 mM to 500 mM.

The above described procedures produce biosynthetic FolliculotropinReleasing Hormone (FRH) free from the known decapeptide hormone LRH. Thebiosynthetic FRH of this invention is readily obtained by theseprocedures and is radioactively labeled with ¹⁴ C for ease ofidentification and as an aid to the purification procedures.

Folliculotropin Releasing Hormone (FRH) is an important regulator ofgonadal function in mammalian species. It could be used separately or inconjunction with the decapeptide LRH. In many respects, FRH may be moreimportant to fertility control than LRH.

The examples given below are provided to exemplify the invention, andmodification of these examples in terms of the sequential uses of thepurification steps, as well as the selection of experimental conditionsas to incubation time, temperature, solvents, column sizes, adsorbents,and other experimental details are all considered in the scope of theinvention. While all of these alternatives are successful, in principle,some of the alternatives are more practical for isolation purposes thanothers.

The following examples are presented to illustrate methods of carryingout the present invention, and it should be understood that incubationmedium reagents, radioactively labeled amino acids, column dimensionsand chromatographic systems are only illustrative and are not intendedto be limitative of the possible means available to produceradioactively labeled biosynthetic Folliculotropin Releasing Hormone(FRH). Additional available buffered incubation media which fall withinthe scope of this invention incude, but are not limited to such buffersas citrate, acetate, formate, carbonate-bicarbonate, Trishydrochloride.Radioactive labeled amino acids include, but are not limited to tritium(³ H) and carbon-14 (¹⁴ C) substitution at any position and any specificactivity commercially available that can be incorporated by thehypothalamic tissue into Folliculotropin Releasing Hormone (FRH). Gelfiltration materials available include, but are not limited to such gelsas Sephadex G-10, G-15 and G-25; and Bio-Gel P-2 and P-4. Partitionchromatography supports include, but are not limited to Sephadex G-25Fine, Bio-Gel P-2 (200-400 mesh) and Sephadex LH-20. Ion exchangechromatographic supports include, but are not limited to such supportsas carboxymethylcellulose (CMC), diethylaminoethylcellulose (DEAE-C),carboxymethyl-Sephadex (CM-S), diethylaminoethyl-Sephadex (DEAE-S),sulfoethyl-Sephadex (SE-S), sulfopropyl-Sephadex (SP-S),diethyl-(2-hydroxy-propyl) aminoethyl-Sephadex (QAE-S),carboxymethyl-Bio-Gel (CM-BG) and BioRad AG-11A8 Ion Retardation resin(BG-AG).

Results of radioactive distribution are graphically represented in thefigures of the accompanying drawing wherein:

FIG. 1 is a graph illustrating the distribution obtained from separationon Bio-Gel P-2 of SP 102;

FIG. 2 is a graph illustrating the distribution obtained from separationon partition on column A of Stage PC-A₂ samples;

FIG. 3 is a graph illustrating the distribution obtained from separationon column B of Stage PC-B samples; and,

FIG. 4 is a graph illustrating the distribution obtained from separationon partition column B of Stage P-2 samples.

EXPERIMENTAL PROCEDURES THE BIOSYNTHESIS AND ISOLATION OF THEFOLLICULOTROPIN RELEASING HORMONE (FRH)

Section a. the biosynthesis of the Folliculotropin Releasing Hormone(FRH).

CHART I; EXAMPLE I

Incubation of Porcine Hypothalamic Fragments to Produce BiosyntheticFolliculotropin Releasing Hormone (FRH)

Porcine hypothalamic fragments were collected at Hormel and Co.slaughterhouse, Austin, Minnesota. They were placed in a Nalgene bottlein batches of 500 fragments and packed in wet ice in a styrofoam box andsent by Air Mail to the Institute for Biomedical Research, Austin,Texas. The ice cold tissue was received the day following shipment.About 25 shipments have been received and incubated. The weight of 500fragments ranged from 213 g to 372 g.

Each batch of 500 hypothalamic fragments was homogenized and incubatedin 500 ml of a medium containing 20 mM potassium phosphate, pH 7.4, 150mM mannitol, 8 mM MgCl, 80 mM KCl, 0.2 mM EDTA, 10 mM sodium-succinate,1 mM ADP, amino acid mixture 0.25 mM in each amino acid, 50 μC ¹⁴C-glutamic acid (UL, specific activity 260 mC/mmole) and 50 μC ¹⁴C-glutamine (UL, specific activity 10 mC/mmole). The amino acid mixturecontained Arg-, His-, Leu-, Ile-, Lys.HCl, Met-, Phe-, Thr-, Trp-, Val-,Ala-, Asp-, Asn-, Glu-, Gln-, Gly-, Hyp-, Pro-, Ser-, Tyr- and Cys.HCl.Incubation was performed for 4 hours at 32°C under an atmosphere of 95%air and 5% carbondioxide. After 4 hours the incubation mixture wasfrozen and lyophilized. The dry, lyophilized, incubated tissue wasstored in a freezer until it was extracted.

Extraction of Biosynthetic Folliculotropin Releasing Hormone (FRH)(Stage SP)

Four lyophilized batches of incubated tissue, equivalent to 2,000fragments were combined for extraction of the biosyntheticFolliculotropin Releasing Hormone. In a few cases, the extraction wasmade on 1,000 or 500 fragment equivalents. In these cases the volumes ofthe solvents used were one-half and one-fourth respectively of that usedfor extraction of 2,000 fragment equivalents.

The 2,000 fragment equivalents were suspended in 1,100 ml cold 15%methanolic acetic acid, and homogenized for 2 minutes in a 1 gallonstainless steel Waring blender. The homogenate was transferred to a 3liter Erlenmeyer flask and the blender was rinsed twice with 100 ml ofmethanolic acetic acid. The homogenate was kept at room temperature for4 hours with occasional shaking before it was filtered under reducedpressure on a large Buchner funnel with a Whatman No. 1 filter paper.The filter cake was washed with 100 ml of methanolic acetic acid andthen suspended in 500 ml of methanolic acetic acid. The suspenpion waskept at room temperature for 2 hours and then in a cold room atapproximately +5°C overnight. It was refiltered and the filter cakeagain washed with 100 ml of 15% HOAc/MeOH.

The combined filtrates were evaporated on a vacuum pump rotaryevaporator, the temperature never exceeding 20°C.

The dry residue was dissolved in 350 ml of boiling distilled water andheated at boiling temperature for about 2 minutes until a clear darkbrown solution was formed. The solution was quickly cooled on ice andextracted 3 times with methylene chloride in a separatory funnel using400, 200 and 100 ml of methylene chloride respectively. The combinedmethylene chloride layer was extracted with 100 ml distilled water. Thecombined aqueous solution was evaporated on a rotary evaporator at about40°C for 1/2 hour to remove dissolved methylenechloride and then frozenand lyophilized. The lyophilized preparation was dissolved with heatingin methanol. The solution was kept in a refrigerator overnight andfiltered. The residue on the filter paper was washed twice with 25 mlmethanol. The combined methanol filtrate was evaporated on a rotaryevaporator and the residue was finally dried on a lyophilizer. Thisresidue constitutes a standard preparation (Stanprep) of theFolliculotropin Releasing Hormone (Stage SP). The weight of fiveStanpreps, each equivalent to 2000 hypothalamic fragments ranged from29.3 g to 41.1 g. Bioassay data on several Stanpreps are in Table I.

Gel Filtration Chromatography of Folliculotropin Releasing HormonePreparations on Bio-Gel P-2 (Stage P-2)

Each Stanprep equivalent to 2,000 hypothalamic fragments waschromatographed on Bio-Gel P-2 100-200 mesh. The gel was swollen ineither 0.2 N or 1.0 N aqueous acetic acid and packed in a column 5.0 ×150 mm. The Stanprep, dissolved in about 50 ml, was applied to andeluted from the column with either 0.2 N or 1.0 N acetic acid. Fractionsof 14 ml were collected. The radioactive distribution in the fractionswas determined and fractions were pooled according to radioactivity,lyophilized, and bioassayed in vitro for their release of luteotropinand folliculotropin. An example of the radioactive distribution from aP-2 column is given in FIG. I. and bioassay data are recorded in Table2. The weight of the FRH active fractions was decreased to 5-10 g, whichis 15-25% of the original weight.

Fractions equivalent to 10,000 hypothalamic fragments, No. 170-300 fromfive P-2 separations, which showed an exceptionally high release offolliculotropin were pooled and passed over a P-2 column a second time.Again the radioactive distribution was determined and fractions werepooled and bioassayed. Bioassay data are given in Table 3. The contentsof tubes 140-400 were most potent in releasing folliculotropin and werechosen for further separation. The weight of these fractions was 19.0 g,which is 11.1% of the original weight of the five Stanpreps (171.5 g).

Separation of the Activities of Biosynthetic FRH and LRH Decapeptide

Many different methods were used for the further purification of FRH andfor separating its activity from that of the luteotropin releasinghormone (LRH). An example is given of a series of continuous separationsof one sample which succeeded in separating the activities of these twohormones. These steps are outlined in Chart I.

Following this, the purification of FRH from other samples with the helpof a diversity of separation methods will be exemplified. (Charts II andIII)

Isolation of Folliculotropin Releasing Hormone Activity, PartitionChromatography on Sephadex G-25 using 0.1% HOAc/H₂ O-nBuOH-Pyr, 11-5-3.System A. (Stage PC-A₁)

Sephadex G-25 fine was swollen in the lower phase of the two phasesolvent system 0.1% aqueous acetic acid-nbutanol-pyridine, 11-5-3, andpacked in a column 2 × 90 cm. The column was washed with about 400 ml ofthe upper phase before a preparation previously purified on two P-2columns, (Stage P-2) equivalent to 1,000 h.f.e. and weighing about 3 gwas applied. The sample was dissolved in a few mls of the lower phase,adsorbed into the top of the column and eluted from the column with theupper phase while 10 ml fractions were collected. The fractions werepooled and assayed both in vivo and in vitro for FRH and LRH activity asshown in Table 4 and Table 5.

The contents of tubes 11-35 was the most active in releasing LH and wasused for isolating the LRH activity on a carboxymethyl cellulose (CMC)column. The contents of tubes 56-100 was used for isolating the FRHactivity.

Rechromatography of Folliculotropin Releasing Hormone (FRH) on aSephadex G-25 Partition Column System A. (Stage PC-A₂)

The column was prepared and the sample from tubes 56-100 (about 1g) ofthe previous column was rechromatographed in the same way as describedabove. The radioactive distribution was determined (FIG. II), thecontents of the tubes were pooled based on this distribution pattern andbioassayed. (Table 6). Two separated fractions, active in release of LHand FH were obtained. The activity in tubes 11-25 corresponds to LRH andis much less than the corresponding activity from the first PC-Aseparation. Most of the LRH activity was removed in the first PC-Aseparation. The activity in tubes 46-112 is essentially unchangedcompared to the bioassay activity from the first separation and thecontents in these tubes were further purified as described below.

Sephadex G-25 Partition Chromatography-Solvent System B:n-Butanol-Acetic-Water, (4-1-5) (Stage PC-B)

Sephadex G-25 fine was swollen in the lower phase of the freshlyprepared two phase solvent system n-butanol-acetic acid-water (4-1-5)and packed in a column 2 × 100 cm. The gel was washed with about 400 mlof the upper phase and the sample from the previous partition separation(PC-A₂) (tubes 46-112, about 0.75 g) was dissolved in the lower phaseand put onto the top of the column. The column was eluted with the upperphase and 10 ml fractions were collected.

Again the radioactive distribution over the tubes was determined and thetubes pooled according to this distribution and bioassayed. (FIG. IIIand Table 7). The best activity for release of FH was found in tubes26-40. The contents of these tubes were submitted to furtherpurification by ion exchange chromatography on DEAE Sephadex.

Ion Exchange Chromatography of Folliculotropin Releasing Hormone on DEAESephadex. (Stage DEAE-S).

The diethylaminoethyl (DEAE) Sephadex was swollen overnight in 1.0 Mammonium acetate (NH₄ OAc)pH 7, filtered under reduced pressure,extensively washed with 0.002 M NH₄ OAc pH 7 and packed in a column 1.5× 30 cm. The necessary amounts of 0.002 M NH₄ OAc, pH 7 and 0.100 M NH₄OAc, pH 4 were mixed to give solutions 0.010 M, 0.020 M, 0.040 M, 0.060M and 0.080 M in NH₄ OAc, the pH also ranging from 7 to 4.

The sample from tubes 26-40 from the previous partition chromatrographicseparation (about 0.3 grams) was dissolved in 0.002 M NH₄ OAc andadsorbed onto the top of the column. The column was eluted with 50 mleach of the stepwise NH₄ OAc gradient solutions described above. Finallythe column was washed with 50 ml of a solution 0.5 M in NH₄ OAc and 2 Min HOAc. All fractions were bioassayed for release of LH and FH. The 0.5M NH₄ OAc eluate showed exceptionally good folliculotropin releasinghormone activity. (Table 8)

Isolation of the Luteotropin Releasing Hormone (LRH) Activity

The luteotropin releasing hormone which is a decapeptide has alreadybeen isolated from hypothalamic tissue and its structure has beenelucidated and confirmed by synthesis. The behavior of this hormone indifferent chromatographic systems is known. The separation of theactivity of LRH and FRH on a second partition chromatography column byuse of 0.1% aqueous HOAc-n-BuOH-Pyr, 11-5-3, has been described (PC-A₂).

A partial purification was obtained already on the first partitionchromatography column (PC-A₁).

The sample in tubes 11-35 from this separation was used for furtherisolation of the LRH activity.

Carboxymethylcellulose Chromatography

Carboxymethylcellulose was packed in a 1.5×30 cm column and equilibratedwith 0.002 M NH₄ OAc pH 4.5. The dry sample was dissolved in the samesolvent and adsorbed onto the top of the column. It was eluted off thecolumn by use of a continuous gradient from 0.002 M NH₄ OAc pH 4.5 to0.100 M NH₄ OAc pH 7. A fraction active for release of both LH and FHwas eluted with approximately 0.07 M NH₄ OAc (Table 9). It has both thechromatrographic and the bioassay characteristics of the LRHdecapeptide.

CHART II: EXAMPLE II

Purification of Folliculotropin Releasing Hormone (FRH) by Use of SomeDifferent Separation Methods

SEPARATION OF FRH FREE FROM LRH

Phenol Extraction

A 2.5 g sample from the first separation on Bio-Gel P-2, dissolved indistilled water, saturated with hydrogen sulfide, was extracted withphenol as described by Schally et al. (Schally et al., Endocrinology,78, 726 (1966)). About 0.5 g was extracted into the phenol phase.Contrary to the findings of Schally et al., the water phase had goodactivity for release of Folliculotropin. Table 10.

Column Chromatographic Separations

1. The sample in the water phase from the phenol extraction (about 2 g)was purified on a 0.1% aqueous HOAc-nBuOH-Pyr, 11-5-3, partition columnin the same way as has been described above. Similar to what has beendescribed above, two well separated fractions, active in release ofLuteotropin and Folliculotropin were obtained Table 11. The first one intubes 1-27 corresponds to the decapeptide, LRH, the second activefraction in tubes 56-102, releases folliculotropin and corresponds toFRH, (Stage PC-A₁).

2. This fraction was further chromatographed on carboxymethylcelluloseby using a continuous ammonium acetate gradient, 0.002 M pH 4.5 to 0.1 MpH 7. Column size 1.5×30 cm.

A fraction very active in release of Folliculotropin was eluted off thecolumn with 0.002 M NH₄ OAc (Stage CMC₁). Table 12. No activity wasfound in fractions corresponding to the behavior of LRH.

SEPARATION OF LRH FREE FROM FRH

Column Chromatography Separation

1. The sample in the phenol phase was next chromatographed on a 0.1%aqueous acetic acid-nBuOH-Pyr, 11-5-3, partition column (2 × 90 cm).Good activity for release of Luteotropin was found in tubes 4-26. Table13. This corresponds to the behavior of LRH (Stage PC-A₂).

2. The sample in tubes 4-26 was submitted to ion exchange chromatographyon a CMC column (1.5 × 30 cm). By using a continuous NH₄ OAc gradient0.002 M pH 4.5 to 0.100 M pH 7, a fraction active in release ofLuteotropin was eluted from the column with about 0.07 M NH₄ OAc Table14. This behavior corresponds to that of LRH (Stage CMC).

CHART III: EXAMPLE III

Purification of Folliculotropin Releasing Hormones (FRH) on a SephadexG-25 Partition Column Using nBuOH-HOAc-H₂ O, 4-1-5, a System B

Samples chromatographed twice on a Bio-Gel P-2 column have been furtherpurified on a Sephadex G-25 partition column (2.5 × 90 cm), by use ofthe solvent system, nBuOH-HOAc-H₂ O, (4-1-5). The column was prepared asdescribed above. Several samples, weighing from 1.5 to 4.85 g have beenfractionated. Between each separation the column was emptied and the gelextensively washed with lower phase. The column was then repacked andwashed with upper phase before being used again.

Fractions of 10 ml were collected. The radioactive distribution in thefractions was determined and the contents of the tubes pooled accordingto this distribution FIG. IV. Assay data on some of these separationsare in Table 15. The FRH activity was found in tubes 31-75 when a 2.5 ×90 cm column was used, and in tubes 21-45 when a 2.0 × 90 cm column wasused. Five separations, making a total of 9,400 h.f.e. (19.0 g) yielded4.2 g of FRH active fractions (Stage PC-B).

Purification of Folliculotropin Releasing Hormone (FRH) on SephadexLH-20.

Sephadex LH-20 was swollen in distilled water overnight and packed intoa column (1.5 × 45 cm). The column was washed with 150 ml of distilledwater-nbutanol (100-9 v/v). A 0.55 g (450 h.f.e.) sample, which had beenpurified twice on a Bio-Gel P-2 column, was fractionated on the column.Water-butanol (100-9 v/v) was used to elute the column, 10 ml fractionswere collected. The best FRH activity was found in tubes 8-10. Table 16.(Stage PC-LH). When the column was equilibrated and eluted withwater-butanol (100-6 v/v) the best FRH activity was recovered in tubes6-8. Table 16.

ASSAY PROCEDURES FOR IN VITRO AND IN VIVO FOLLICULOTROPIN RELEASINGHORMONE (FRH) AND LUTEOTROPIN RELEASING HORMONE (LRH) ASSAYS.

For the in vitro assays, pituitaries were obtained from female rats ofthe Sprague-Dawley strain which were 20 days old. Two pituitaries foreach assay were incubated at 37° C in 1 ml of lactated Ringer's solution(Travenol Laboratories) in 10-ml Teflon beakers in a Dubnoff shaker.After a pre-incubation period of 1 hour (PI₁), the medium was removedfor a control assay and fresh medium was added to the system. After asecond hour of pre-incubation (PI₂), the medium was removed for assayand replaced. During the incubation (I), the medium was removed andassayed for release of pituitary hormones four times at one-hourperiods, I₃, I₄, I₅, I₆. The samples to be assayed were added after thetwo-hour pre-incubation period; the total experimental time was 6 hours.LH was determined by the radioimmunoassay method of Niswender et al.(Niswender, et al., Proc. Soc. Exp. Biol. Med., 128, 807 (1968)), andFSH was determined according to Daane and Parlow (Daane and Parlow,Endocrinology, 88, 653 (1971)). The release of LH and FSH were evidentby comparison of the pre-incubation and incubation values. The reagentsfor assay of FSH were generously distributed by Dr. A. Parlow of NIAMD,NIH; Dr. G. Niswender generously supplied the anti-ovine LH serum No.15. Dr. L. E. Reichert, Jr. supplied the ovine LH preparation forlabelling and the reference standard of LH from the rat. The values forthese assays were calculated in terms of ng of the following referencestandards: LH-LER-1240-2 (0.60 NIH-LH-S1 units/mg) and FSH 2.1 ×NIH-FSH-S 1 units/mg.

The in vivo studies were performed in Sprague-Dawley adult female andmale rats. Female rats were ovariectomized 2 or 3 months before theassays. All steroids were administered as a single injection,subcutaneously, 72 hours before injection of the decapeptide. Underether anesthesia, blood was collected from the jugular vein and thedecapeptide was injected into the same vein. Serum assays for LH and FSHwere performed in duplicate by the double antibody radioimmunoassay ofNiswender et al. and Daane and Parlow, respectively. The LH results areexpressed in terms of ng/ml of LER 1240-2-0.6 NIH-LH-S-I units/mg andFSH in terms of a preparation with a potency of 2.1 × NIH-FSH-S-Iunits/mg.

Section b. the isolation of the Folliculotropin Releasing Hormone

CHART IV; EXAMPLE IV.

Extraction of Tissue For Standard Preparation (Stanprep) ofFolliculotropin Releasing Hormone (FRH)

Fresh porcine hypothalamic tissues were placed in Nalgene bottles at theslaughter house. Each bottle contained 500 hypothalamic fragmentequivalents (h.f.e.) in 400 ml of reagent grade methanol. The Nalgenebottles of hypothalami were shipped by air mail and were placed underrefrigeration upon arrival.

The cold hypothalamic fragments in batches of 5,000 h.f.e. werecollected by filtration through a large Buchner funnel without filterpaper. The partially dried glands were then lyophilized. The decantedfiltrate was saved for later use. The lyophilized tissue (about500g/5,000 h.f.e.) is thoroughly homogenized in 4,000 ml of cold 15% byweight glacial acetic acid in methanol using a 1 gallon stainless steelWaring blender. The homogenate was allowed to stand for at least 1 hourin the cold room before filtration. The homogenate was filtered using alarge Buchner funnel and Whatman No. 1 filter paper under reducedpressure. The filter cake was resuspended in 15% acetic acid in methanoland refiltered after at least 2 hours standing in the cold room. Thisprocedure was repeated until a total of 5 filtrations had beenaccomplished. The resultant combined filtrates were evaporated todryness under reduced pressure and added to the decanted filtrate fromthe tissue. The entire methanolic extract was evaporated to dryness.

The resultant dark brown, syrupy residue was dissolved in 350 to 400 mlof 2 M acetic acid (aqueous) and extracted three times with 400 ml ofmethylene chloride (CH₂ Cl₂). The combined CH₂ Cl₂ layer was backwashedwith 150 ml of 2 M acetic acid and then extracted with 150 ml of CH₂Cl₂. The combined aqueous layers were then concentrated to dryness usinga vacuum pump. The syrupy residue was taken up in about 300 ml ofmethanol and cooled to 0°C overnight. The resulting fine precipitate wasremoved by filtration and the filtrate evaporated to dryness to give 50g of "Stanprep" having Folliculotropin Releasing Hormone (FRH) activityfrom 5000 h.f.e. The in vitro assay results are shown in Table 17.

Gel Filtration Chromatography of Folliculotropin Releasing Hormone onBio-Gel P-2

Batches (50 g each) of "Stanprep" were dissolved in 100 ml of 1 M aceticacid and applied to a Bio-Gel P-2 column (5.0 × 135 cm) (100-200 mesh)(V₀ = 910ml) (Exclusion limit 1800 Daltons) that was previouslyequilibrated in the same solvent. The column was eluted using a 2-3 ft.pressure head of solvent and 14 ml fractions were collected. Appropriatefractions were pooled and an aliquot from each pooled fraction wassubmitted for biological assay. The assay results of the pooledfractions are shown in Table 18. As shown in Table 18. theFolliculotropin Releasing Activity (FRH) activity is located inFractions No. 101-250. In general the gel filtration fractions aredivided into four major groups:

                            LRH and                                                                 Wt. % FRH Activity %                                        ______________________________________                                        Group A = No. 1-100                                                                              Inactive 2.1   0                                           "     B = No. 101-175                                                                            Active   84.5  40-45                                       "     C = No. 176-300                                                                            Active   13.1  55-60                                       "     D = No. 301-500                                                                            Inactive .3    0                                           ______________________________________                                    

Group B fractions are then rechromatographed on the same Bio-Gel P-2column described above. A similar weight and hormonal activitydistribution is obtained.

    ______________________________________                                                     Wt. %  LRH and FRH Activity %                                    ______________________________________                                        Group BA = No. 1-100                                                                             2.3      0                                                 "     BB = 101-175 90.3     40-45                                             "     BC = 176-300 7.1      55-60                                             "     BD = 301-500 0.3      0                                                 ______________________________________                                    

Group C fractions are combined from two identical columns, 10,000h.f.e., and rechromatographed on Bio-Gel P-2. The in vitro assay data isshown in Table 19.

The activity and weight distribution is as shown:

                 Wt. %  LRH and FRH Activity %                                    ______________________________________                                        Group CA = No. 1-100                                                                             0.7      0                                                 "     CB = 101-175 73.8     6                                                 "     CC = 176-275 25.2     94                                                "     CD = 276-500 0.3      0                                                 ______________________________________                                    

Group BC fractions are combined from two or three identical columns andrechromatographed to give similar results to those with combined Group Cfractions. The in vitro assay data are shown in Table 20.

Folliculotropin Releasing Hormone (FRH) Partition Chromatography SystemA: n- Butanol-Pyridine-0.1% Acetic Acid (5:3:11).

A column (5.0 × 55 cm) was packed with Sephadex G-25 Fine that had beenswollen overnight in the lower phase of the solvent systemn-Butanol-Pyridine-0.1% acetic acid (5:3:11). The interstitial solventthat is excluded from the gel particles was replaced with the upperphase of solvent system A. A sample, 6.57g, was dissolved in 15 ml ofthe lower phase and applied to the top of the column. The column waseluted with the upper phase and 12 ml fractions were collected. AfterFraction No. 300, the solvent was changed to lower phase of System A.The assay data is shown in Table 21. The activity and weightdistribution is as shown below.

    ______________________________________                                                           Activity %                                                               Wt. %  LRH       FRH                                            ______________________________________                                        Group A   21-100    19.0     85      0                                        Group B  101-140    14.9     15      <5                                       Group C  141-300    53.1     0       80                                       Group D  301-380    13.0     0       <20                                      ______________________________________                                    

For smaller samples (3-4 g), a column (2.0 × 85 cm) packed in the samemanner as above was used. The Folliculotropin Releasing Hormone (FRH)and Luteotropin Releasing Hormone (LRH) activity distribution is shownin Table 22 and the weight distribution is as shown for 10 ml fractions:

                       Activity %                                                               Wt. %  LRH       FRH                                            ______________________________________                                        Group A  11-30      4.5      85      0                                        Group B  31-40      5.8      15      <5                                       Group C  41-80      57.5     0       95                                       Group D   81-120    32.2     0       <5                                       ______________________________________                                    

Folliculotropin Releasing Hormone (FRH): Partition Chromatography SystemB: n-Butanol-Acetic acid-Water (4:1:5)

A column (2.5 × 80 cm) was packed with Sephadex G-25 Fine swollen in thelower phase of the solvent system: n-Butanol-Acetic acid-Water (4:1:5).The interstitial solvent was then displaced with the upper phase. Asample of 3.67g from Partition Column A (FRH region) was dissolved in 5ml of lower phase and applied to the top of the column. The column waseluted with the upper phase while collecting 10 ml fractions. TheFolliculotropin Releasing Hormone (FRH) activity is shown in Table 23.The weight distribution is as shown below:

                   Wt. %   FRH Activity %                                         ______________________________________                                        Group I   11-40      14.0      0                                              Group II  41-60      11.8      50                                             Group III 61-90      7.8       0                                              Group IV   91-110    66.4      50                                             ______________________________________                                    

Folliculotropin Releasing Hormone (FRH): CarboxymethylcelluloseChromatography

A column (1.5 × 80 cm) was packed with carboxymethyl cellulose that hadbeen prepared by the method of Roy and Konigsberg (Roy and Konigsberg,Methods in Enzymology, 25, 221 (1972)) and then equilibrated with 2 mMpH 4.5 NH₄ OAc. A sample, 2.03 g, of FRH from Partition Column System Awas dissolved in 10 ml of starting buffer and applied to the column. Alinear gradient to 100 m M, pH 7 NH₄ OAc was started at fraction No. 11and fractions of 10 ml were collected. A small amount of LRH activitypresent in the starting material appears in Fraction No. 61-65 (70 m M).The FRH activity (1.62 g) was unretained and was eluted in Fractions No.6-10. The assay results are shown in Table 24.

CHART V; EXAMPLE V.

Folliculotropin Releasing Hormone (FRH) Chromatography on Bio-RadAG-11A8 Ion Retardation Resin

A sample of 2.25 g of FRH from Partition Column System A was dissolvedin 15 ml of water and applied to a column (1.5 × 80 cm) of Bio-RadAG-11A8 (50-100 mesh) resin. The column was eluted with water whilecollecting 10 ml fractions and finally eluted with 1 M acetic acid. TheFRH assay results, in vitro, are shown in Table 25. The weight andactivity distribution is as shown:

                       Activity %                                                               Wt. %  LRH       FRH                                            ______________________________________                                        Group I  1-20       74.2     100     25                                       Group II 21-40      8.5      0       0                                        Group III                                                                              41-70      8.6      0       0                                        Group IV 71-165     1.1      0       15                                       Group V  1 M HOAc   7.6      0       60                                       ______________________________________                                    

Diethylaminoethylcellulose Chromatography

A column (1.5 × 25 cm) was packed with DEAE cellulose prepared by themethod of Roy and Konigsberg. The column was equilibrated with 2 m M, pH9, NH₄ OAc and a sample of 0.69 g of FRH from Partition Column A anddesalting on Bio-Rad AG-11A8. The column was eluted with a continuouslinear gradient buffer to pH 7, 100 m M NH₄ OAc. The column was thenwashed with 2 M HOAc. The activity and weight distribution is shownbelow. In vitro assay results of pooled fractions (5 ml) are in Table26.

    ______________________________________                                                        Wt. %    Activity %                                           ______________________________________                                        Group I    6-10       72.4       <5                                           Group II  11-70       20.2       0                                            Group III 71-110      2.8        0                                            Group IV  2 M HOAc    4.3        <95                                          ______________________________________                                    

Folliculotropin Releasing Hormone (FRH): Cellulose Thin LayerChromatography

A sample of FRH from Partition Column System A-Ion Retardation resinGroup I was divided and chromatographed on two identical Cellulose TLCplates. One plate was developed in Chloroform-Methanol-Aqueous NH₄ OH(60:45:20) and the second plate was developed in Chloroform-Methanol-32%Aqueous Acetic acid (60:45:20). The FRH activity is shown in Table 27.

    CHART I EXAMPLE I                                                             __________________________________________________________________________    Batches of 500 Fresh Porcine Hypothalami                                              ↓ Buffered Medium                                                      ↓ Amino Acid Mixture                                                   ↓ ATP Synthesizing System                                              ↓ .sup.14 C-glutamic acid and .sup.14 C-glutamine                      ↓ 32°C, 95%Air/5%CO.sub.2, 4 hours                              Stage SP. Folliculotropin Releasing Hormone (FRH) and                         Luteotropic Releasing Hormone (LRH) Labeled with .sup.14 C.                   ↓ BioGel P-2                                                           ↓ 0.2M or 1.0M HOAc                                                    Stage P2 FRH + LRH                                                            ↓ Partition Chromatography                                             ↓ System A                                                             CM-cellulose                                                          Stage PC-A.sub.1                                                                      FRH + Stage PC-A.sub.1 LRH →Stage CMC LRH                              Linear Gradient                                                               ↓ Partition Chromatography                                             ↓ System A                                                     Stage PC-A.sub.2                                                                      FRH + Stage PC-A.sub.2 LRH                                                    ↓ Partition Chromatography                                             ↓ System B                                                     Stage PC-B                                                                            FRH                                                                           ↓ DEAE-Sephadex                                                        ↓ Stepwise Gradient                                            Stage DEAE-S                                                                          FRH                                                                   __________________________________________________________________________

    CHART II EXAMPLE II                                                           __________________________________________________________________________    Stage P2 Folliculotropin Releasing Hormone (FRH) and                          Luteotropic Releasing Hormone (LRH). Labeled with .sup.14 C.                                      Phenol extraction                                         H.sub.2 O                                                                           Phase                PhOH                                                                              Phase                                          ↓                                                                            Partition Chromatography                                                                           ↓                                                                          Partition Chromatography                       ↓                                                                            System A             ↓                                                                          System A                                       Stage PC-A FRH + Stage PC-A LRH                                                                          Stage                                                                             PC-A LRH                                       ↓                                                                            Carboxy methyl cellulose                                                                           ↓                                                                           Carboxy methyl cellulose                      ↓                                                                            Chromatography       ↓                                                                          Chromatography                                 ↓                                                                            Linear gradient      ↓                                                                          Linear gradient                                Stage CMC FRH              Stage                                                                             CMC LRH                                        __________________________________________________________________________

    CHART III EXAMPLE III                                                         ______________________________________                                        Stage P2 Folliculotropin Releasing Hormone (FRH)                                       Labeled with .sup.14 C.                                              Partition              Partition                                              Chromatography         Chromatography                                         System B               Sephadex LH-20                                         Stage PC-B FRH          Stage PC-LH FRH                                       ______________________________________                                    

    CHART IV EXAMPLE IV                                                           __________________________________________________________________________    Batches of 5000 Lyophilized Hypothalami                                       ↓               Methanolic acetic acid                                                        Extraction                                                                    Methylene chloride defatting                           Stage SP               Folliculotropin Releasing                              Hormone                (FRH) and Luteotropin Releasing                        Hormone                (LRH)                                                  ↓               BioGel P-2                                                                    1.0M HOAc                                              Stage P-2              FRH and LRH                                            ↓               Partition Chromatography                                                      System A                                                       CMC                     CMC                                           Stage CMC FRH                                                                         ←                                                                             Stage PC-A FRH +                                                                        Stage PC-A LRH                                                                         →                                                                           Stage CMC LRH                                    linear                  linear                                                or                      or                                                    stepwise                stepwise                                              gradient                gradient                                                   ↓  Partition Chromatography                                                      System B                                                            Stage     PC-B FRH                                                            ↓  DEAE-Sephadex or                                                              DEAE-Cellulose                                                                Linear or Stepwise Gradient                                         Stage     DEAE-S FRH                                             __________________________________________________________________________

    CHART V EXAMPLE V                                                             ______________________________________                                                 Stage PC-A FRH                                                                    Bio Rad AG-11A8                                                               Ion Retardation                                                               Resin, H.sub.2 O then HOAc                                                 Stage AG FRH                                                        cellulose           DEAE-Cellulose                                              TLC               Linear Gradient                                           Stage TLC FRH           Stage DEAE-C FRH                                      ______________________________________                                    

                                      Table 1                                     __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH)                     and Luteotropin Releasing Hormone (LRH) in Stanpreps                          Stage SP)                                                                                            ng FSH/ml                                              IBR Descrip-   Preincubation                                                                         Incubation                                             No. tion h.f.e.                                                                              P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                            I.sub.4                                                                            I.sub.5                                                                             I.sub.6                                __________________________________________________________________________    2933                                                                              SP 101                                                                             0.6, 3.0                                                                            1000                                                                              <1000                                                                             12,500                                                                             52,000                                                                             60,000                                                                              52,000                                 3163                                                                              SP 102                                                                             0.5, 7.5                                                                            1250                                                                              <1000                                                                              3,250                                                                             38,750                                                                             >128,000                                                                            46,250                                 3388                                                                              SP 105                                                                             0.5, 2.5                                                                            --  1800                                                                              --   17,750                                                                             51,750                                                                              --                                     __________________________________________________________________________                           ng LH/ml                                               __________________________________________________________________________    2933                                                                              SP 101                                                                             0.6, 3.0                                                                            20  20  378  >714 >714  >714                                   3163                                                                              SP 102                                                                             0.5, 7.5                                                                            20  38  165  >714 >714  >714                                   __________________________________________________________________________

                                      Table 2                                     __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH)                     and Luteotropin Releasing Hormone (LRH) in Samples From                       First Bio-Gel P-2 Separation (Stage P-2)                                                               ng FSH/ml                                            IBR Descrip-   Preincubation                                                                           Incubation                                           No. tion h.f.e.                                                                              P.sub.1                                                                            P.sub.2                                                                            I.sub.3                                                                             I.sub.4                                                                            I.sub.5                                                                             I.sub.6                             __________________________________________________________________________    2910                                                                              SP 101                                                                             0.2, 3                                                                              <1000                                                                              < 1000                                                                              9000 10,750                                                                             42,000                                                                              68,000                              2915                                                                              SP 101                                                                             0.2, 3                                                                              1000 < 1000                                                                             26,000                                                                              37,500                                                                             126,000                                                                             51,000                              3165                                                                              SP 102                                                                             0.2, 2                                                                              2000 2750 17,150                                                                              13,500                                                                             52,500                                                                              >128,000                            3167                                                                              SP 102                                                                             0.2, 2                                                                              2300 2650 28,000                                                                              34,000                                                                             67,500                                                                              43,250                              3168                                                                              SP 102                                                                             0.2, 2                                                                              53000                                                                              4300 10,150                                                                               8,900                                                                             >128,000                                                                            64,750                              3276                                                                              SP 104                                                                             0.2, 1                                                                              3000 3500 20,000                                                                              --   76,750                                                                              68,500                              3277                                                                              SP 104                                                                             0.2, 1                                                                              5000 5000 110,000                                                                             --   68,500                                                                              54,000                              3278                                                                              SP 104                                                                             0.2, 1                                                                              5000 4500 64,000                                                                              --   59,500                                                                              39,250                              3392                                                                              SP 105                                                                              0.1, 0.5                                                                           --   1650 --    12,650                                                                             12,250                                                                              --                                  3393                                                                              SP 105                                                                              0.1, 0.5                                                                           --   1900 --    13,500                                                                             16,500                                                                              --                                  3394                                                                              SP 105                                                                              0.1, 0.5                                                                           --   5150 --     5,050                                                                              7,600                                                                              --                                  __________________________________________________________________________                             ng LH/ml                                             __________________________________________________________________________    2910                                                                              SP 101                                                                             0.2, 3                                                                              14   13   151   168  >714  >714                                2915                                                                              SP 101                                                                             0.2, 3                                                                              20   20   131   528  >714  >714                                3165                                                                              SP 102                                                                             0.2, 2                                                                              50   60   155   258  >714  >714                                3167                                                                              SP 102                                                                             0.2, 2                                                                              63   50   375   662  >714  >714                                3168                                                                              SP 102                                                                             0.2, 2                                                                              225  88    80   115  >714  >714                                3276                                                                              SP 104                                                                             0.2, 1                                                                              25   29   215   490  >714  >714                                3277                                                                              SP 104                                                                             0.2, 1                                                                              126  208  405   683  >714  >714                                3278                                                                              SP 104                                                                             0.2, 1                                                                              294  148  692   714  >714  >714                                __________________________________________________________________________

                                      Table 3                                     __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone                           (FRH) in Pooled Samples From Second Bio-Gel P-2                               Separation (Stage P-2)                                                                         ng FSH/ml                                                    IBR Descrip-     Preincubation                                                                         Incubation                                           No. tion    h.f.e.                                                                             P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                         I.sub.4                                                                            I.sub.5                                                                            I.sub.6                                  __________________________________________________________________________    3432                                                                              IBR 101-105                                                                           0.2, 1                                                                             --  2000                                                                              --                                                                              8100 25,700                                                                             --                                       3433                                                                              "       "    --  1750                                                                              --                                                                              19100                                                                              34,650                                                                             --                                       3434                                                                              "       "    --  1250                                                                              --                                                                              11400                                                                              29,950                                                                             --                                       3435                                                                              "       "    --  2250                                                                              --                                                                              12550                                                                              15,600                                                                             --                                       3436                                                                              "       "    --  3500                                                                              --                                                                              8250 19.950                                                                             --                                       3437                                                                              "       "    --  2500                                                                              --                                                                              9800 14,600                                                                             --                                       3438                                                                              "       "    --  3100                                                                              --                                                                              9100 43,250                                                                             --                                       __________________________________________________________________________

                  Table 4                                                         ______________________________________                                        In Vivo Assay of Luteotropin Releasing Hormone                                (LRH) in Samples from Stage PC-A.sub.1                                        IBR  Description                                                                              Tubes     h.f.e.                                                                              Before After                                  ______________________________________                                        2748 May 5      11-35     3     7.2    224                                                                    12     127                                    2749 May 5      36-55     3     12     25.2                                                                   9.2    12                                     7750 May 5      56-75     3     15.6   12.6                                                                   7.2    39                                     7751 May 5       76-100   3     7.6    12                                                                     8      96                                     ______________________________________                                    

                                      Table 5                                     __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormones                          (FRH) and Luteotropin Releasing Hormone (LRH) in                              Samples from Stage PC-A.sub.1                                                                    ng FSH/ml                                                  IBR Descrip-       Preincubation                                                                         Incubation                                         No. tion Tubes h.f.e.                                                                            P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                             I.sub.4                                                                             I.sub.5                                                                            I.sub.6                           __________________________________________________________________________    2976                                                                              May 5                                                                              36-55 1,5 2400                                                                              1250                                                                                2,250                                                                             17,100                                                                              56,500                                                                             51,150                            2977                                                                              May 5                                                                              56-75 1,5 2000                                                                              1900                                                                              121,000                                                                             112,500                                                                             38,000                                                                             30,750                            2978                                                                              May 5                                                                               76-100                                                                             1,5 1000                                                                              1000                                                                               55,500                                                                             46,750                                                                              40,500                                                                             16,600                            __________________________________________________________________________                               ng LH/ml                                           __________________________________________________________________________    2976                                                                              May 5                                                                              36-55 1,5 78  58   115   340  > 714                                                                              > 714                             2977                                                                              May 5                                                                              56-75 1,5 138 50  > 714 > 714 > 714                                                                              > 714                             2978                                                                              May 5                                                                               26-100                                                                             1,5 30  40  > 714 > 714 > 714                                                                              > 714                             __________________________________________________________________________

                                      Table 6                                     __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH) and                 Luteotropin Releasing Hormone (LRH) in Samples from Stage PC-A.sub.2                             ng FSH/ml                                                  IBR Descrip-       Preincubation                                                                          Incubation                                        No. tion Tubes h.f.e.                                                                            P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                            I.sub.4                                                                           I.sub.5                                                                            I.sub.6                              __________________________________________________________________________    3213                                                                              May 5                                                                              11-25 1,5 5000                                                                              4000                                                                              8,000                                                                              --  17,500                                                                             18,000                               3214                                                                              May 5                                                                              26-45 1,5 3000                                                                              5000                                                                              4,000                                                                              --   4,500                                                                             5,500                                3215                                                                              May 5                                                                              46-65 1,5 3500                                                                              5000                                                                              52,000                                                                             --  40,000                                                                             28,250                               3216                                                                              May 5                                                                              66-85 1,5 3300                                                                              4500                                                                              33,000                                                                             --  67,000                                                                             47,500                               3217                                                                              May 5                                                                               86-112                                                                             1,5 4000                                                                              4000                                                                              5,000                                                                              --  25,000                                                                             55,500                               __________________________________________________________________________                               ng LH/ml                                           __________________________________________________________________________    3213                                                                              May 5                                                                              11-25 1,5 94  79  179  168 460  555                                  3214                                                                              May 5                                                                              26-45 1,5 25  10   18   14  29   41                                  3215                                                                              May 5                                                                              46-65 1,5 201 142 622  717 > 714                                                                              >714                                 3216                                                                              May 5                                                                              66-85 1,5 59  69  358  535 > 714                                                                              >714                                 3217                                                                              May 5                                                                               86-112                                                                             1,5 39  48   30  123 243  617                                  __________________________________________________________________________

                                      Table 7                                     __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone in                        Samples from Stage PC-B                                                                          ng FSH/ml                                                  IBR Descrip-       Preincubation                                                                         Incubation                                         No. tion Tubes                                                                              h.f.e.                                                                             P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                         I.sub.4                                                                            I.sub.5                                                                            I.sub.6                                __________________________________________________________________________    3421                                                                              May 5                                                                               5-25                                                                              2,10 --  1500                                                                              --                                                                              4,900                                                                              8,650                                                                              --                                     3422                                                                              May 5                                                                              26-40                                                                              2,10 --  2000                                                                              --                                                                              33,250                                                                             43,500                                                                             --                                     3423                                                                              May 5                                                                              41-50                                                                              2,10 --  2000                                                                              --                                                                              3,650                                                                              5,000                                                                              --                                     3425                                                                              May 5                                                                              51-70                                                                              2,10 --  1250                                                                              --                                                                              2,500                                                                              4,750                                                                              --                                     __________________________________________________________________________

                                      Table 8                                     __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH)                     and Luteotropin Releasing Hormone (LRH) in Samples from                       Stage DEAE-S.                                                                                 ng FSH/ml                                                     IBR             Preincubation                                                                         Incubation                                            No. Description                                                                          Fraction                                                                           P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                         I.sub.4                                                                           I.sub.5                                                                            I.sub.6                                    __________________________________________________________________________    3652                                                                              May 5  0.04 M                                                                             --  2400                                                                              --                                                                              6500                                                                              3,550                                                                              --                                         3653                                                                              May 5  0.06 M                                                                             --  1900                                                                              --                                                                              4700                                                                              3,600                                                                              --                                         3654                                                                              May 5  0.08 M                                                                             --  2600                                                                              --                                                                              6400                                                                              4,300                                                                              --                                         3656                                                                              May 5  0.5  M                                                                             --  2600                                                                              --                                                                              --  90,750                                                                             --                                         __________________________________________________________________________                            ng LH/ml                                              __________________________________________________________________________    3656                                                                              May 5  0.5  M                                                                             --   130                                                                              --                                                                              445 > 714                                                                              --                                         __________________________________________________________________________

                                      Table 9                                     __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH) and                 Luteotropin Releasing Hormone (LRH) in Samples from Stage                     CMC.                                                                                           ng FSH/ml                                                    IBR              Preincubation                                                                         Incubation                                           No. Description                                                                          Fraction                                                                            P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                            I.sub.4                                                                            I.sub.5                                                                            I.sub.6                               __________________________________________________________________________    2981                                                                              May 5  0.002 M                                                                             2250                                                                              2600                                                                              9,600                                                                              15,500                                                                             19,250                                                                             22,750                                2982                                                                              May 5  0.01  M                                                                             1000                                                                              1350                                                                              7,950                                                                              5,250                                                                              11,000                                                                             14,150                                2983                                                                              May 5  0.04  M                                                                             1900                                                                              2500                                                                              7,700                                                                              3,750                                                                              12,000                                                                             13,000                                2984                                                                              May 5  0.07  M                                                                             3050                                                                              1850                                                                              12,250                                                                             19,500                                                                             30,750                                                                             34,250                                2985                                                                              May 5  0.100 M                                                                             1000                                                                              2000                                                                              2,850                                                                              4,500                                                                               6,000                                                                              6,500                                __________________________________________________________________________                             ng LH/ml                                             __________________________________________________________________________    2981                                                                              May 5  0.002 M                                                                             20  40  165  296  > 714                                                                              560                                   2982                                                                              May 5  0.01  M                                                                             30  39  228  150  348  515                                   2983                                                                              May 5  0.04  M                                                                             30  44  157   85  398  365                                   2984                                                                              May 5  0.07  M                                                                             121 50  283  > 714                                                                              > 714                                                                              > 714                                 2985                                                                              May 5  0.100 M                                                                             32  41   80  125  208  113                                   __________________________________________________________________________

                  Table 10                                                        ______________________________________                                        In Vivo Assay of Folliculotropin Releasing Hormone (FRH) and                  Luteotropin Releasing Hormone (LRH) in Samples from Phenol                    Extraction of Stage P-2 Preparations.                                                                ng FSH/ml                                              IBR No.  Description Phase   h.f.e.                                                                              Before                                                                              After                                ______________________________________                                        2338     Jan. 20     PhOH    0.5   3330  2800                                                                    3210  2890                                 2339     Jan. 20     H.sub.2 O                                                                             5     4260  4990                                                                    3400  4680                                 ______________________________________                                                               ng LH/ml                                               ______________________________________                                        2338     Jan. 20     PhOH    0.5    6    25                                                                       4    27                                   2338     Jan. 20     H.sub.2 O                                                                             5     16    74                                                                      12    79                                   ______________________________________                                    

                  Table 11                                                        ______________________________________                                        In Vivo Assay of                                                              Folliculotropin Releasing Hormone (FRH) and                                   Luteotropin Releasing Hormone (LRH) in Samples                                from Stage PC-A                                                                                     ng FSH/ml                                               IBR No.  Description Fraction   Before After                                  ______________________________________                                        2602     Jan. 20      1-27      3800   3300                                   2604     Jan. 20     56-80      2850   2950                                                                   2850   2800                                   2605     Jan. 20      81-102    2350   3300                                                                   1800   2000                                   ______________________________________                                                              ng LH/ml                                                ______________________________________                                        2602     Jan. 20      1-27      14     62                                                                     14     43                                     2604     Jan. 20     56-80      14     33                                                                      8     50                                     2605     Jan. 20      81-102    < 4    242                                                                     6     23                                     ______________________________________                                    

                                      Table 12                                    __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH) and                 Luteotropin Releasing Hormone (LRH) in Samples from Stage                     CMC.sub.1                                                                                      ng FSH/ml                                                    IBR              Preincubation                                                                          Incubation                                          No. Description                                                                          Fraction                                                                            P.sub.1                                                                            P.sub.2                                                                           I.sub.3                                                                            I.sub.4                                                                          I.sub.5                                                                            I.sub.6                                __________________________________________________________________________    2704                                                                              Jan. 20                                                                              0.002 M                                                                             < 1000                                                                             2100                                                                              10250                                                                              1900                                                                             55,500                                                                             59,500                                 2705                                                                              Jan. 20                                                                              0.01 M                                                                              2400 3950                                                                              12000                                                                              8750                                                                             28,500                                                                             28,500                                 2706                                                                              Jan. 20                                                                              0.04 M                                                                              1050 2000                                                                              2500 2350                                                                             4,000                                                                              3,750                                  2707                                                                              Jan. 20                                                                              0.07 M                                                                              1000 1800                                                                              2300 2150                                                                             3,250                                                                              3,750                                  2708                                                                              Jan. 20                                                                              0.10 M                                                                              1250 2050                                                                              2200 3300                                                                             3,500                                                                              3,250                                  __________________________________________________________________________                              ng LH/ml                                            __________________________________________________________________________    2704                                                                              Jan. 20                                                                              0.002 M                                                                             < 10 10  260  525 > 714                                                                              > 714                                 2705                                                                              Jan. 20                                                                              0.01 M                                                                              65   20  340  415 > 714                                                                              582                                   2706                                                                              Jan. 20                                                                              0.04 M                                                                              20   20  40   53   68   38                                   2707                                                                              Jan. 20                                                                              0.07 M                                                                              26   20  50   35   60   40                                   2708                                                                              Jan. 20                                                                              0.10 M                                                                              10   10  18   50   38   70                                   __________________________________________________________________________

                  Table 13                                                        ______________________________________                                        In Vivo Assay of                                                              Folliculotropin Releasing Hormone (FRH) and                                   Luteotropin Releasing Hormone (LRH) in Samples                                from Stage PC-A.sub.2                                                                              ng FSH/ml                                                IBR    Description  Fraction   Before  After                                  ______________________________________                                        2340   Jan. 20       4-26      3200    3830                                                                  5300    5160                                   ______________________________________                                                             ng LH/ml                                                 ______________________________________                                        2340   Jan. 20       4-26       4      >285                                                                  10       263                                   2341   Jan. 20      27-40      14      16                                                                    <4      <4                                     2342   Jan. 20      41-55      12      15                                                                    10      11                                     2559   Jan. 20      56-80       6       8                                                                    <4      <4                                     2560   Jan. 20       81-100     4       4                                                                    <4      15                                     ______________________________________                                    

                  Table 14                                                        ______________________________________                                        In Vivo Assay of                                                              Luteotropin Releasing Hormone (LRH) in                                        Samples from Stage CMC                                                                             ng LH/ml                                                 IBR     Description Fraction   Before  After                                  ______________________________________                                        2564    Jan. 20     0.002 M    <4      <4                                                                    6       6                                      2565    Jan. 20     0.01 M     <4      24.5                                                                  6       12                                     2566    Jan. 20     0.02 M     10      14                                                                    12      14                                     2567    Jan. 20     0.04 M     6       6                                                                     8       6.4                                    2568    Jan. 20     0.05 M     7       8                                                                     4       4                                      2569    Jan. 20     0.07 M     4       54.2                                                                  4       72.0                                   2570    Jan. 20     0.10 M     4       4                                                                     4       4                                      ______________________________________                                    

                                      Table 15                                    __________________________________________________________________________    In Vitro Assay of                                                             Folliculotropin Releasing Hormone (FRH) in                                    Samples from Stage PC-B                                                                       ng FSH/ml                                                     IBR             Preincubation                                                                          Incubation                                           No. Description                                                                          Fraction                                                                           P.sub.1                                                                            P.sub.2                                                                           I.sub.3                                                                          I.sub.4                                                                            I.sub.5                                                                            I.sub.6                                 __________________________________________________________________________    3753                                                                              SP101-105                                                                            24-40                                                                              --   3250        50,500                                                                             --                                      3794                                                                              SP101-105                                                                            21-45                                                                              --   2600                                                                              -- 46,000                                                                             43,000                                                                             --                                      3797                                                                              SP101-105                                                                            36-75                                                                              --   2600                                                                              -- 51,000                                                                             41,250                                                                             --                                      __________________________________________________________________________

                                      Table 16                                    __________________________________________________________________________    In Vitro Assay of                                                             Folliculotropin Releasing Hormone (FRH) in                                    Samples from Stage PC-LH                                                                      ng FSH/ml                                                     IBR             Preincubation                                                                          Incubation                                           No. Description                                                                          Fraction                                                                           P.sub.1                                                                            P.sub.2                                                                           I.sub.3                                                                          I.sub.4                                                                          I.sub.5                                                                            I.sub.6                                   __________________________________________________________________________    3747                                                                              SP101-105                                                                            8-10 --   3350                                                                              -- -- 54500                                                                              --                                             9% BuOH                                                                  3733                                                                              SP101-105                                                                            6-8  --   3150                                                                              -- -- 46700                                                                              --                                              6% BuOH                                                                 __________________________________________________________________________

                                      Table 17                                    __________________________________________________________________________    In Vitro Assay of Folliculotropin                                             Releasing Hormone (FRH) in Stanprep (Stage SP)                                               ng FSH/ml                                                      IBR Descrip-                                                                           Wt. of                                                                              Preincubation                                                                         Incubation                                             No. tion dose, mg                                                                            P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                            I.sub.4                                                                            I.sub.5                                                                            I.sub.6                                 __________________________________________________________________________    3093                                                                              DH-133                                                                             3.7,18.7                                                                            1450                                                                              3600                                                                              31,500                                                                             51,500                                                                             52,500                                                                             39,000                                  3023                                                                              FK-I 5.6,29.1                                                                            1000                                                                              1500                                                                              13,750                                                                             18,900                                                                             46,000                                                                             >128,000                                3173                                                                              FK-II                                                                              3.9,19.4                                                                            2850                                                                              3600                                                                              20,600                                                                             51,500                                                                             54,750                                                                             26,250                                  3777                                                                              FK-III                                                                               27.5                                                                              --  2300                                                                              --   --   35,500                                                                             --                                      __________________________________________________________________________

                                      Table 18                                    __________________________________________________________________________    In Vitro Assay of                                                             Folliculotropin Releasing Hormone (FRH) from                                  Pooled Fractions After Bio-Gel P-2 Chromatography                             of 5000 h.f.e. (Stage P-2)                                                                ng FSH/ml                                                         IBR Descrip-                                                                              Preincubation                                                                          Incubation                                               No. tion    P.sub.1                                                                            P.sub.2                                                                           I.sub.3                                                                            I.sub.4                                                                              I.sub.5                                                                              I.sub.6                               __________________________________________________________________________    3025                                                                              FK-I-101-                                                                             2400 2300                                                                               4650                                                                               3750   17,250                                                                              35,750                                    125                                                                       3026                                                                              FK-I-126-                                                                             2400 3600                                                                              38,500                                                                             104,500                                                                              >128,000                                                                             40,250                                    150                                                                       3027                                                                              FK-I-151-                                                                             4000 3550                                                                              58,250                                                                             >128,000                                                                             >128,000                                                                             53,500                                    175                                                                       3028                                                                              FK-I-176-                                                                             4050 4250                                                                              14,500                                                                             >128,000                                                                             >128,000                                                                             49,000                                    200                                                                       3029                                                                              FM-I-201-                                                                             2800 2500                                                                              36,750                                                                             92,500 >128,000                                                                             >128,000                                  225                                                                       3030                                                                              FK-I-226-                                                                             3500 3350                                                                              10,000                                                                             18,150 38,650 34,000                                    250                                                                       3095                                                                              DH-125-101-                                                                           7300 8500                                                                              24,500                                                                             16,600 16,150 19,750                                    125                                                                       3096                                                                              DH-125-126-                                                                           9350 7000                                                                              33,500                                                                             35,000 32,500 41,000                                    150                                                                       3097                                                                              DH-125-151-                                                                           <1000                                                                              1600                                                                              40,500                                                                             51,750 38,500 24,000                                    175                                                                       3098                                                                              DH-125-176-                                                                           2050 4000                                                                              49,500                                                                             39,000 22,100 21,250                                    200                                                                       3099                                                                              DH-125-201-                                                                           6000 8000                                                                              25,250                                                                             22,750 49,000 39,000                                    250                                                                       2995                                                                              DH-128-180-                                                                           3600 3250                                                                              33,750                                                                             58,000 >128,000                                                                             100,000                                   359                                                                       __________________________________________________________________________

                                      Table 19                                    __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH) from                Pooled Fractions After Rechromatography on Bio-Gel P-2 of 10,000 h.f.e.       (No. 180-360) (Stage P-2)                                                                   ng FSH/ml                                                                     Preincubation                                                                          Incubation                                             IBR No.                                                                            Description                                                                            P.sub.1                                                                           P.sub.2                                                                            I.sub.3                                                                            I.sub.4                                                                            I.sub.5                                                                            I.sub.6                                 __________________________________________________________________________    2997 DH-128-101-                                                                            1950                                                                              3500 14,500                                                                             22,750                                                                             39,500                                                                             30,250                                        125                                                                     2998 DH-128-126-                                                                            2150                                                                              4000 10,350                                                                             10,750                                                                             10,000                                                                             13,000                                        150                                                                     2999 DH-128-151-                                                                            2050                                                                              4000 5750 5750 19,750                                                                             38,750                                        175                                                                     3000 DH-128-176-                                                                            1100                                                                              3000 61,000                                                                             76,500                                                                             76,500                                                                             32,250                                        200                                                                     3001 DH-128-201-                                                                            2850                                                                              3250 40,500                                                                             73,500                                                                             83,000                                                                             40,000                                        225                                                                     3003 DH-128-226-                                                                            1100                                                                              1000  5,600                                                                              7,100                                                                             43,500                                                                             43,750                                        250                                                                     3456 DH-129-2-106-                                                                          --  2750 --   22,000                                                                             47,750                                                                             --                                            175                                                                     3457 DH-129-2-176-                                                                          --  2250 --   23,000                                                                             27,650                                                                             --                                            275                                                                     3515 DH-129-3-101-                                                                          --  <1000                                                                              --   17,256                                                                             36,500                                                                             --                                            175                                                                     3516 DH-129-3-176-                                                                          --  <1000                                                                              --   37,250                                                                             27,250                                                                             --                                            300                                                                     __________________________________________________________________________

                  Table 20                                                        ______________________________________                                        In Vitro Assay of Folliculotropin Releasing Hormone (FRH)                     Fractions After Rechromatography of Group B Fractions From                    First P-2 (Stage P-2)                                                         ______________________________________                                                  ng FSH/ml                                                           IBR   Descrip-  Preincubation                                                                              Incubation                                       No.   tion      P.sub.1                                                                             P.sub.2                                                                              I.sub.3                                                                           I.sub.4                                                                              I.sub.5                                                                              I.sub.6                        ______________________________________                                        3518  FK-II-                                                                        101-175   --    <1000  --  45,500 43,000 --                             3519  FK-II-                                                                        176-300   --    <1000  --  56,250 50,500 --                             3602  FK-I-                                                                         101-175   --    4150   --  65,250 28,000 --                             3603  FK-I-                                                                         176-300   --    2350   --  66,750 30,150 --                             3605  DH-115-                                                                       101-175   --    3550   --  87,000 71,750 --                             3606  DH-115-                                                                       176-319   --    4900   --  51,900 81,500 --                             ______________________________________                                    

                                      Table 21                                    __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH) of                  Fractions from Partition Column: System A. (5.0×55 cm column)           (Stage PC-A)                                                                                  ng FSH/ml                                                     IBR Descrip-                                                                            Wt. of                                                                              Preincubation                                                                         Incubation                                            No. tion  dose, mg                                                                            P.sub.1                                                                         P.sub.2                                                                             I.sub.3                                                                         I.sub.4                                                                            I.sub.5                                                                            I.sub.6                                   __________________________________________________________________________    3554                                                                              DH-129-                                                                             0.1,0.5                                                                             --                                                                              1300  --                                                                              6,500                                                                              16,950                                                                             --                                            21-40                                                                     3528                                                                              DH-129-                                                                             0.4,1.8                                                                 41-60       --                                                                              1350  --                                                                              14,350                                                                             28,250                                                                             --                                        3529                                                                              DH-129-                                                                             1.6,8.3                                                                 81-100      --                                                                              1650  --                                                                              2,300                                                                              21,500                                                                             --                                        3530                                                                              DH-129-                                                                             1.1,5.8                                                                 101-120                                                                             120     --    <1000                                                                           --   <1,000                                                                             5,500                                                                           --                                      3531                                                                              DH-129-                                                                             1.1,5.4                                                                 121-140     --                                                                              1250  --                                                                              6,300                                                                              25,300                                                                             --                                        3532                                                                              DH-129-                                                                             0.7,3.5                                                                 141-160       2600  --                                                                              15,800                                                                             62,500                                                                             --                                        3533                                                                              DH-129-                                                                             1.6,8.3                                                                 161-180     --                                                                              1000  --                                                                              46,900                                                                             45,750                                                                             --                                        3534                                                                              DH-129-                                                                             2.1,10.6                                                                181-200     --                                                                              <1000 --                                                                              42,600                                                                             36,900                                                                             --                                        3535                                                                              DH-129-                                                                             1.4,6.9                                                                 201-220     --                                                                              <1000 --                                                                              41,500                                                                             37,250                                                                             --                                        3536                                                                              DH-129-                                                                             0.8,4.1                                                                 221-240     --                                                                              1850  --                                                                              27,300                                                                             56,750                                                                             --                                        3537                                                                              DH-129-                                                                             0.5,2.3                                                                 261-280     --                                                                              1350  --                                                                              14,750                                                                             58,000                                                                             --                                        3538                                                                              DH-129-                                                                             0.3,1.5                                                                 301-320     --                                                                              <1000 --                                                                              1,500                                                                              18,000                                                                             --                                        3635                                                                              DH-129-                                                                             1.4,5.8                                                                 321-340     --                                                                              2450  --                                                                              18,750                                                                             69,500                                                                             --                                        3636                                                                              DH-129-                                                                             0.7,3.4                                                                 341-360     --                                                                              2200  --                                                                              9,750                                                                              37,500                                                                             --                                        3637                                                                              DH-129-                                                                             0.1,0.6                                                                 361-380     --                                                                              1300  --                                                                              4,250                                                                              3,000                                                                              --                                    

                                      Table 22                                    __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone (FRH)                     in Fractions from Partition Column. System A. (2.0×85 column)           (Stage PC-A)                                                                                  ng FSH/ml                                                     IBR Descrip-                                                                            Wt. of                                                                              Preincubation                                                                           Incubation                                          No. tion  dose, mg                                                                            P.sub.1                                                                            P.sub.2                                                                            I.sub.3                                                                            I.sub.4                                                                            I.sub.5                                                                            I.sub.6                              __________________________________________________________________________    3076                                                                              DH-128-                                                                       51-70       <1000                                                                              1550 39,500                                                                             34,000                                                                             53,500                                                                             41,250                               3079                                                                              DH-128-                                                                       101-120     1000 <1000                                                                              1,000                                                                              1,100                                                                              2,600                                                                              3,650                                3116                                                                              DH-128-                                                                       141-160     4100 4500 4,000                                                                              4,000                                                                              3,500                                                                              2,650                                3117                                                                              BC-57-                                                                        11-20       3700 5250 16,750                                                                             12,500                                                                             19,750                                                                             4,750                                3118                                                                              BC-57-                                                                        21-30       5500 5750 22,500                                                                             4,750                                                                              14,750                                                                             16,250                               3119                                                                              BC-57-                                                                        31-40       2500 4750 5,100                                                                              6,250                                                                              9,150                                                                              8,850                                3120                                                                              BC-57-                                                                        41-50       3350 3150 27,000                                                                             37,500                                                                             49,500                                                                             46,500                               3121                                                                              BC-57                                                                         51-60       3500 3500 33,800                                                                             51,000                                                                             42,000                                                                             30,000                               3122                                                                              BC-57                                                                         61-70       1950 1550 39,500                                                                             63,100                                                                             49,500                                                                             40,000                               3147                                                                              BC-57                                                                         71-80       2100 3050 4,000                                                                              6,600                                                                              25,250                                                                             36,250                               3148                                                                              BC-57                                                                         81-90       3100 3650 1,750                                                                              1,350                                                                              2,500                                                                              3,250                                3149                                                                              BC-57                                                                         90-100      5740 4950 3,000                                                                              3,300                                                                              2,200                                                                              2,750                                3152                                                                              BC-57-2-                                                                      21-30       1000 1400 2,350                                                                              3,100                                                                              2,500                                                                              2,750                                3153                                                                              BC-57-2-                                                                      31-40       1200 1600 2,000                                                                              1,750                                                                              3,000                                                                              1,900                                3154                                                                              BC-57-2-                                                                      41-45       4450 5250 4,500                                                                              5,000                                                                              6,750                                                                              3,000                                3155                                                                              BC-57-2-                                                                      46-55       1050 1300 39,900                                                                             48,500                                                                             45,000                                                                             30,000                               3760                                                                              BC-63-                                                                        21-30 0.1,0.5                                                                             --   3050 --   --   3,300                                                                              --                                   3761                                                                              BC-63-                                                                        31-40 0.2,1.1                                                                             --   4550 --   --   4,400                                                                              --                                   3762                                                                              BC-63-                                                                        41-50 0.5,2.4                                                                             --   3400 --   --   9,250                                                                              --                                   3814                                                                              BC-63-                                                                        51-60 0.7,3.7                                                                             --   3000 --   24,000                                                                             69,500                                                                             --                                   3815                                                                              BC-63-                                                                        61-70 0.4,2.1                                                                             --   3000 --   2,500                                                                              4,000                                                                              --                                   3763                                                                              BC-63-                                                                        71-80 0.3,1.5                                                                             --   2650 --   --   2,250                                                                              --                                   3764                                                                              BC-63-                                                                        81-113                                                                              0.8,4.0                                                                             --   4050 --   --   5,500                                                                              --                                   __________________________________________________________________________

                                      Table 23                                    __________________________________________________________________________    In Vitro Assay of Folliculotropin Releasing Hormone                           (FRH in Fraction from Partition Column System B. (2.5×80 cm)            (Stage PC-B)                                                                                       ng FSH/ml                                                                Wt. of                                                                             Preincubation                                                                         Incubation                                       IBR No.                                                                            Description                                                                              dose,mg                                                                            P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                         I.sub.4                                                                         I.sub.5                                                                            I.sub.6                                 __________________________________________________________________________    3765 DH-129-A-11-25  --  3650                                                                              --                                                                              --                                                                              2950 --                                      3766 "    26-40      --  4350                                                                              --                                                                              --                                                                              3600 --                                      3767 "    41-50 2.3  --  4800                                                                              --                                                                              --                                                                              22,000                                                                             --                                      3768 "    51-60 2.0  --  2950                                                                              --                                                                              --                                                                              46,150                                                                             --                                      3769 "    61-70      --  3800                                                                              --                                                                              --                                                                              1500 --                                      3770 "     91-100    --  4500                                                                              --                                                                              --                                                                              33,750                                                                             --                                      3771 "    101-110                                                                             24.1 --  2750                                                                              --                                                                              --                                                                              53,750                                                                             --                                      3893 "    111-120                                                             3896 "    121-130                                                             3889 "    131-190                                                             __________________________________________________________________________

                                      Table 24                                    __________________________________________________________________________    In Vitro Folliculotropin Releasing Hormone (FRH)                              Activity of Fractions from Carboxymethylcellulose                             Chromatography (Stage CMC)                                                                    ng FSH/ml                                                     IBR Descrip-                                                                            Wt. of                                                                              Preincubation                                                                         Incubation                                            No. tion  dose, mg                                                                            P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                            I.sub.4                                                                            I.sub.5                                                                             I.sub.6                               __________________________________________________________________________    3039                                                                              DH-128-                                                                       A-No. 6                                                                             0.5,2.6                                                                             6400                                                                              4200                                                                              17,650                                                                             12,750                                                                             48,500                                                                              48,000                                3040                                                                              DH-128-                                                                       A-No. 7                                                                             0.5,2.4                                                                             4000                                                                              4900                                                                              12,600                                                                             23,500                                                                             80,500                                                                              92,500                                3041                                                                              DH-128-                                                                       A-No. 8                                                                             0.4,2.0                                                                             2800                                                                              4350                                                                              14,550                                                                             20,000                                                                             103,500                                                                             99,000                                3042                                                                              DH-128-                                                                       A-No. 9                                                                             0.4,1.8                                                                             2900                                                                              3850                                                                              18,950                                                                             34,750                                                                             >128,000                                                                            >128,000                              3043                                                                              DH-128-                                                                       A-No. 10                                                                            0.2,1.0                                                                             3100                                                                              3200                                                                              4,500                                                                              4,650                                                                              8,550 9,500                                 3062                                                                              DH-128-                                                                       A-No.61-65  3350                                                                              3150                                                                              3,850                                                                              3,850                                                                              9,850 17,300                                __________________________________________________________________________

                                      Table 25                                    __________________________________________________________________________    In Vitro FRH Activity of Fractions From Bio-Rad AG-11A8                       Ion Retardation Resin (Stage AG)                                                               ng FSH/ml                                                    IBR        Wt. of                                                                              Preincubation                                                                         Incubation                                           No. Description                                                                          dose, mg                                                                            P.sub.1                                                                           P.sub.2                                                                           I.sub.3                                                                         I.sub.4                                                                            I.sub.5                                                                            I.sub.6                                  __________________________________________________________________________    3640                                                                              DH-128-PC-                                                                    A: 1-20                                                                              1.0,5.1                                                                             --  1100                                                                              --                                                                              6700 24,850                                                                             --                                       3461                                                                              DH-128-PC-                                                                    A: 21-40                                                                             0.2,1.1                                                                             --  3000                                                                              --                                                                              2750  5,300                                                                             --                                       3462                                                                              DH-128-PC                                                                     A: 41-70                                                                             0.3,1.6                                                                             --  1700                                                                              --                                                                              3150  2,650                                                                             --                                       3463                                                                              DH-128-PC-                                                                    A: 71-165                                                                            0.4,2.0                                                                             --  3750                                                                              --                                                                              2300 16,350                                                                             --                                       3611                                                                              DH-128-PC-                                                                    A: 1M HOAc                                                                           2.0,11.0                                                                            --  2250                                                                              --                                                                              39,750                                                                             99,500                                                                             --                                       __________________________________________________________________________

                                      Table 26                                    __________________________________________________________________________    In Vitro FRH Activity of Fractions From Diethylaminoethyl                     cellulose Chromatography (Stage DEAE)                                                           ng FSH/ml                                                   IBR  Descrip-                                                                            Wt. of Preincubation                                                                           Incubation                                        No.  tion  Dose, mg                                                                             P   P.sub.2                                                                             I.sub.3                                                                           I.sub.4                                                                            I.sub.5                                                                            I.sub.6                             __________________________________________________________________________    3309 BC-57-2-                                                                      6-10  0.6,3.0                                                                              1750                                                                              <1000 4400                                                                              3150 4350 5500                                3310-                                                                              BC-57-2-                                                                 3317 11-70 0.2,1.1                                                                              1860                                                                              1370  2290                                                                              2540 2720 1980                                3318-                                                                              BC-57-2-                                                                 3321 71-110                                                                              0.02,0.12                                                                            1990                                                                              1590  2050                                                                              3210 2500 2750                                3612 BC-57-2-                                                                      2 M HOAc                                                                            0.06,0.3                                                                             --  1500  --  17,500                                                                             66,750                                   __________________________________________________________________________

                                      Table 27                                    __________________________________________________________________________    In Vitro Folliculotropin Releasing Hormone (FRH) Activity After               Cellulose TLC in Basic and Acidic Solvent Systems (Stage TLC)                                  ng FSH/ml                                                    IBR Descrip-                                                                             Wt. of                                                                              Preincubation                                                                           Incubation                                         No. tion   dose, mg                                                                            P.sub.1                                                                             P.sub.2                                                                           I.sub.3                                                                           I.sub.4                                                                            I.sub.5                                                                            I.sub.6                              __________________________________________________________________________        BASIC                                                                     3323                                                                              BC-57-AG-                                                                     Band 1       1300  1300                                                                              2750                                                                              1000 3000 2400                                 3324                                                                              BC-57-AG-                                                                     Band 2       1150  1100                                                                              3000                                                                              2000 2050 3000                                 3325                                                                              BC-57-AG-                                                                     Band 3 0.4,2.0                                                                             1100  2100                                                                              5400                                                                              5750 24,500                                                                             26,200                               3326                                                                              BC-57-AG-                                                                     Band 4       2100  1100                                                                              2100                                                                              1500 1800 3350                                 3327                                                                              BC-57-AG-                                                                     Band 5       <1000 1050                                                                              3000                                                                              2050 3800 3600                                 3328                                                                              BC-57-AG-                                                                     Band 6       2150  1450                                                                              1100                                                                              2500 3650 3400                                     ACIDIC                                                                    3704                                                                               "Band 1     --    3300                                                                              --  --   7500 --                                   3705                                                                               "Band 2     --    3300                                                                              --  --   8500 --                                   3706                                                                               "Band 3     --    2350                                                                              --  --   6500 --                                   3707                                                                               "Band 4                                                                  3708                                                                               "Band 5                                                                  3709                                                                               "Band 6                                                                  3710                                                                               "Band 7     --    2000                                                                              --  14,200                                                                             42,650                                                                             --                                   3711                                                                               "Band 8     --    2400                                                                              --  4350 5250 --                                   __________________________________________________________________________

What is claimed:
 1. The new and partially isolated hypothalamic hormone,Folliculotropin Releasing Hormone (FRH), which fulfills the samebiological function as the intact hypothalamus of mammalians ofreleasing folliculotropin and which has an Rf of about 0.4 on celluloseTLC in the solvent system chloroform-methanol-32% acetic acid (60:45:20)and an Rf of about 0.6 on cellulose TLC in the solvent systemchloroform-methanol-29% ammonium hydroxide (60:45:20).
 2. A method forbiosynthetically preparing the Folliculotropin Releasing Hormone (FRH)to provide an increased yield at high purity of said FRH directly fromhypothalamic tissue said method comprising lyophilizing incubatedhypothalamic tissue and thereafter extracting and defatting saidlyophilized tissue to obtain a standard preparation; twice passing saidstandard preparation through a filtering medium; chromatographing saidtwice passed preparation a first time on a partition column in a solventsystem which is selected from those systems consisting of 0.1% aceticacid in water-n-butanol-pyridine (11-5-3); separating said preparationby means of a linear or step-wise gradient into those components whichexhibit LRH and FRH activities; and, chromatographing a second time thatseparated component which exhibits FRH activity in said solvent systemto biosynthetically obtain FRH.
 3. A method for diagnosing and analysingfertility in mammals comprising administering to a mammal a biologicallyeffective amount of the FRH hormone of claim
 1. 4. The method of claim 2wherein said method includes identifying said biosynthetically producedFRH by tagging its amino acids with a radioactive substance selectedfrom the group consisting of tritium and carbon-14.